The molecular pathogenesis of cancer and other diseases is related to genetic changes, such as mutation or altered gene expression. For example, genetic changes in tumors can be correlated to cancer metastasis, treatment outcome, and survival using modern high-throughput molecular biologic and proteomic methods. This biological information permits the development and selection of therapeutic agents targeted to specific molecular alterations in the tumor. When the clinical course is short, as in pancreatic cancer, the molecular information can be obtained using fresh or frozen tissue from which intact mRNA and protein is easily extracted. However, for cancers that have a longer clinical course (such as breast and prostate cancer) many years may elapse between initial diagnosis or treatment and the appearance of metastases. In these instances, clinical correlations are obtained using formalin-fixed paraffin-embedded (FFPE) tissues. Unfortunately, analysis of rare mRNA transcripts by RT-PCR from FFPE tissue is currently unreliable. Large-scale multiplex techniques (such as proteomic analysis, serial analysis of gene expression, and gene chip methods) using FFPE tissue have therefore been unreliable.
Most archival tissues have been “fixed” by soaking them in a solution of 3.7% (w/v) formaldehyde, dehydrating them through a series of water/alcohol solutions and xylene, and then embedding them in paraffin wax. This procedure facilitates morphologic examination by preserving cellular integrity. The major method by which formaldehyde achieves this goal is by inactivation of nucleases, proteases, lipases, and saccharidases that cause cells and tissues to “self-destruct” after death or excision.
Attempts have been made to develop fixatives that better preserve tissue for genomic and proteomic analysis, but they are not widely used. Thus, most tissue archives consist of specimens that have been fixed with formaldehyde, dehydrated, and embedded in paraffin wax. Although formaldehyde reaction chemistry has been widely investigated, little attention has been given to studying the processes of dehydration and paraffin embedding that follow fixation, but precede microscopic examination. The understanding of the chemical processes that occur when tissue is prepared for microscopic examination is thus incomplete, which has frustrated the development of methods by which this process could be sufficiently reversed to yield macromolecules suitable for molecular analysis, such as proteomic analysis.
Formaldehyde fixes proteins in tissue by cross-linking basic amino acids, such as lysine and glutamine, and through the formation of methylol adducts with these basic amino acids. Both intra-molecular and inter-molecular cross-links are formed. These cross-links preserve protein secondary structure while destroying enzyme activity by forming active-site adducts, which prevent enzyme conformational changes and inhibit diffusion of both enzyme and substrate through the cellular matrix. Formaldehyde reacts with uncharged amino groups (such as the ε-amino group of Lys) to form highly reactive methylol compounds by the reaction:R—NH2+CH2OR—NH—CH2—OHUnder suitable steric conditions, the reactive methylol compounds condense with amide, phenol, indole, and imidazole side chains to form methylene bridges that cross-link polypeptide chains by reactions such as:R—NH—CH2—OH+NH2—CO—R′R—NH—CH2—NH—CO—R′+H2OThese reactions have discouraged investigators from using FFPE archival tissues for proteomic analysis. Two-dimensional (2-D) gel electrophoresis requires that protein molecules not “stick” to each other, because they must be solubilized to be loaded on the gel and separated by molecular weight during electrophoresis. Furthermore, 2-D separation requires protein ionization for successful isoelectric focusing. The formation of either methylol adducts or methylene cross-links neutralizes basic amines, which significantly perturbs the isoelectric focusing step.
Another unwanted effect of formaldehyde fixation is a reduction of immunohistochemical reactivity in tissue sections. This loss of reactivity is believed to arise from chemical epitope modification and the inability of antibodies to diffuse into the cross-linked tissue. These effects may be partially reversed by exposure of fixed tissue sections to high temperatures for short periods of time in the presence of aqueous salt or protein denaturant solutions. Shi et al. (J. Histochem. Cytochem. 39:741-748, 1991) have demonstrated that heat treatment can improve retrieval of antigens masked by formalin fixation and that optimal results are correlated with the product of the heating temperature and the time of heat treatment (CAP Today 9:116-123, 1995).
The pH of the antigen retrieval (AR) solution has also been found to play a role in staining patterns of fixed tissue. Three distinct staining patterns have been identified: (a) optimal staining at low and high pH; (b) optimal staining correlated with increasing pH; and (c) optimal staining independent of pH (Shi et al., J. Histochem. Cytochem. 43:193-201, 1995). A variety of salt and protein denaturant solutions have been employed for AR; however, no single chemical component has been shown to be universally required or optimal for antigen unmasking (Shi et al., J. Histochem. Cytochem. 45:327-343, 1997).
Shi et al. (J. Histochem. Cytochem. 40:787-792, 1992) have also proposed that the unwanted modification of protein structure by formalin is reversed by treatment with an antigen retrieval (AR) buffer at room temperature.
Processing tissue for histological examination is time consuming, often requiring up to 24 hours to complete. This processing period delays the examination of tissue biopsies by pathologists. In the case of surgical pathology, a surgeon may be waiting for a diagnosis to decide how to proceed, and this delay is costly and can cause harm to the patient. To avoid this problem, rapidly prepared frozen tissue sections are used in surgical pathology, however, such tissue sections lack the anatomic detail of conventional FFPE tissue sections.
Others have recognized the need to shorten the time required for tissue processing, but they have made only modest improvements in the conventional methods. To accelerate tissue processing, U.S. Pat. Nos. 4,656,047, 4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos. 3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No. 5,023,187 uses infrared energy. U.S. Patent Publication 2008/0153127 discloses a method of rapid fixation of tissue specimens at a temperature of 30-65° C. under sub-atmospheric pressure.